Studies show that different strains of Staphylococcus aureus are resistant to a wide variety of antibiotics including vancomycin. The present study was performed to isolate vancomycin resistant strain of S. aureus from clinical specimens and to understand how they acquire the resistance gene in vivo. A total of 38 S. aureus isolates from clinical specimens were tested against vancomycin for antibiotic sensitivity by disk diffusion and tube dilution method. The MIC of vancomycin for these isolates ranged between 16 µg/ml and 1024 µg/ml. The MBC value ranged between 128 µg/ml and 1024 µg/ml. One of the 38 identified isolates showed resistance against vancomycin (MIC of 64 µg/ml and MBC 128 µg/ml). The vancomycin resistant operon vanA was characterized in the Vancomycin resistant S. aureus strain. The resistant isolate was further inoculated into hartleyÂÂs guinea pigs along with a susceptible strain to facilitate conjugal transfer. Samples drawn from the animals at regular intervals were further analyzed to determine antibiotic susceptibility and detection of resistant gene. The molecular examination of the genomic DNA of the S. aureus isolated recombinant strain revealed that all the new strains have acquired vanA operon in vivo essential for antibiotic resistance to S. aureus.